1. Field of the Invention
The invention relates to non-covalent, readily dissociable conjugates between biologically active molecules. More particularly, the invention relates to such conjugates between biologically-active molecules using a flexible nucleic acid linking moiety that is covalently linked to each of the biologically-active molecules and that forms the conjugate by hybridization between the nucleic acid strands. Specifically, the invention relates to such conjugates wherein single-stranded nucleic acids are covalently linked to each of the biologically active molecules, and the conjugate is formed by hybridization of complementary segments of these single-stranded nucleic acids to one another or to a third, separate and distinct, single-stranded or partially single-stranded linking nucleic acid. Methods for making the conjugates of the invention are provided. Also provided are methods for using the conjugates of the invention in chemical or biochemical analysis, purification or manipulation of molecules for research or industrial purposes, or medical diagnosis or treatment.
2. Summary of the Related Art
In the biological and medical arts, it is frequently desirable to link together different molecules of the same or different type, for the performance of an assay or to embody the properties of each of a multiplicity of different molecules in the same conjugate molecule. It has become commonplace to covalently link together two protein molecules or a protein molecule with a non-protein molecule, so that the resulting combined molecule preserves at least some of the chemical properties of each of the original component molecules. Examples are the combination of an antibody molecule with an enzyme, or with a radioactive, or fluorescent, or electron-opaque molecule, so that the site where the antibody binds can be readily detected or the binding properties of the antibody can be more readily exploited for chemical assay of the substance bound by it. In other applications, for example, the antibody may be bound to a toxin, so that when the combined molecule is taken up by a target cell the latter may thereby be damaged or killed.
European Patent Application, Publication No. EP 0 134 127 A2 discloses polyamino acid-based linkers for conjugating proteins (such as antibodies and enzymes) and proteins to solid phases.
Although nucleic acids have been used in the past to interlink protein molecules, the methods employed have not fully exploited the advantages of nucleic acids as interlinking means: for example, the raw materials are readily available, and nucleic acids are of low intrinsic toxicity and antigenicity.
European Patent Application, Publication No. EP 0 154 884 A2 discloses nucleic acid-protein adducts comprising antibody molecules. The nucleic acid is derivatized with hapten, fluorescent or radioactive labels and covalently linked to S. aureus Protein A, and the resulting nucleic acid/antibody conjugates are used to amplify the signal provided by antibody/antigen binding.
International Patent Application, Publication No. WO91/17442 discloses nucleic acid-antibody hybrids comprising a bacteriophage promoter element operatively linked to a transcribed sequence, used to amplify and detect antibody binding to antigen.
European Patent Application, Publication No. EP 0 488 152 A2, discloses an immunoassay method wherein a detectable antibody-antigen complex is connected to a substrate by a double-stranded nucleic acid, which complex is released from the substrate by restriction endonuclease cleavage of the double-stranded nucleic acid.
European Patent Application, Publication No. EP 0 490 434 A1 discloses randomly-placed, multiply derivatized antibody molecules using nucleic acids and evidence that these adducts form duplexes with complementary, labelled nucleic acids.
International Patent Application, Publication No. WO 92/12164 discloses formation of three-dimensional nucleic acid structures for conjugation to proteins and other molecules.
Asseline et al., 1992, Tetrahedron 48: 1233-54 disclose nucleic acid-based heterobifunctional linkers.
Scouten and Konecny, 1992, Analytical Biochemistry 205: 313-8 disclose conjugation of antibodies with magnetic particles via duplex formation between oligo-A and oligo-T nucleic acid chains.
The art does not disclose a readily dissociable, nucleic acid-based linker for conjugating biologically active molecules. Such means have a number of uses. For example, molecules having binding specificities of varying affinity (such as antibodies) can be combined, permitting low-affinity molecules, especially those having great specificity, to be used in assays where their low affinity would otherwise preclude their use. Such linked antibody molecules are envisioned to be useful in improving the performance of immunoassay and competitive binding assays. Dissociable conjugation also permits linkage of targeting molecules (such as antibodies) and cytotoxic or other drugs, thereby improving drug delivery and specificity. In addition, the same or related cytotoxic drugs can be prepared with differing specificities (e.g., for a particular tumor type, targeted with a cell surface-specific targeting molecule); specific drugs could be prepared for an individual patient, based on expression of a particular cell-surface antigen. Finally, extremely high affinity and high selectivity binding conjugates can be made for chemical or biological purification by the combination of specific binding molecules recognizing, e.g., non-overlapping epitopes on a desirable analyte. Such selected and purified analytes can be released by dissociation of the complex, thereby reducing the affinity of binding overall and allowing the analyte to be recovered.